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Elevated post-ischemic ubiquitination results from suppression of deubiquitinase activity and not proteasome inhibition

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Abstract

Cerebral ischemia–reperfusion increases intraneuronal levels of ubiquitinated proteins, but the factors driving ubiquitination and whether it results from altered proteostasis remain unclear. To address these questions, we used in vivo and in vitro models of cerebral ischemia–reperfusion, in which hippocampal slices were transiently deprived of oxygen and glucose to simulate ischemia followed by reperfusion, or the middle cerebral artery was temporarily occluded in mice. We found that post-ischemic ubiquitination results from two key steps: restoration of ATP at reperfusion, which allows initiation of protein ubiquitination, and free radical production, which, in the presence of sufficient ATP, increases ubiquitination above pre-ischemic levels. Surprisingly, free radicals did not augment ubiquitination through inhibition of the proteasome as previously believed. Although reduced proteasomal activity was detected after ischemia, this was neither caused by free radicals nor sufficient in magnitude to induce appreciable accumulation of proteasomal target proteins or ubiquitin–proteasome reporters. Instead, we found that ischemia-derived free radicals inhibit deubiquitinases, a class of proteases that cleaves ubiquitin chains from proteins, which was sufficient to elevate ubiquitination after ischemia. Our data provide evidence that free radical-dependent deubiquitinase inactivation rather than proteasomal inhibition drives ubiquitination following ischemia–reperfusion, and as such call for a reevaluation of the mechanisms of post-ischemic ubiquitination, previously attributed to altered proteostasis. Since deubiquitinase inhibition is considered an endogenous neuroprotective mechanism to shield proteins from oxidative damage, modulation of deubiquitinase activity may be of therapeutic value to maintain protein integrity after an ischemic insult.

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Acknowledgements

The authors wish to thank Diego Santisteban Vargas for technical assistance with ubiquitin-AMC experiments.

Funding

This study was supported by National Institute of Health (NIH) grants R01-NS109588 to KH, R01-NS34179 to CI, and R01-NS067078 to PZ, as well as a post-doctoral research grant from the Deutsche Forschungsgemeinschaft (KA2279/4-1) to TK. KH is the Finbar and Marianne Kenny Research Scholar in Neurology at Weill Cornell Medicine. Support from the Feil Family Foundation is gratefully acknowledged.

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Authors and Affiliations

  1. Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, 10065, USA

    Timo Kahles, Carrie Poon, Liping Qian, Victoria Palfini, Shanmukha Priya Srinivasan, Shilpa Swaminathan, Ismary Blanco, Reunet Rodney-Sandy, Costantino Iadecola, Ping Zhou & Karin Hochrainer

  2. Department of Neurology, Cantonal Hospital Aarau, 5001, Aarau, Switzerland

    Timo Kahles

Authors
  1. Timo Kahles
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  2. Carrie Poon
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  3. Liping Qian
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  4. Victoria Palfini
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  5. Shanmukha Priya Srinivasan
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  6. Shilpa Swaminathan
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  7. Ismary Blanco
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  8. Reunet Rodney-Sandy
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  9. Costantino Iadecola
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  10. Ping Zhou
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  11. Karin Hochrainer
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Contributions

TK, LQ, PZ, and KH conducted brain slice and neuron experiments and performed data analysis; CP, VP, and KH performed MCAO experiments, associated histology and data analysis; SPS contributed to DUB activity experiments; SS aided with proteasome experiments; IB, VP, RRS, and SPS contributed to Western blotting experiments; PZ, CI, and KH provided funding; KH and CI supervised the research and wrote the manuscript.

Corresponding author

Correspondence to Karin Hochrainer.

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Conflict of interest

CI is on the Scientific Advisory Board of Broadview Ventures. The other authors declare that they have no competing interests.

Ethics approval

All procedures involving mice were approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee (IACUC) and were executed according to IACUC, NIH, and ARRIVE guidelines (https://www.nc3rs.org/ARRIVE).

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Kahles, T., Poon, C., Qian, L. et al. Elevated post-ischemic ubiquitination results from suppression of deubiquitinase activity and not proteasome inhibition. Cell. Mol. Life Sci. 78, 2169–2183 (2021). https://doi.org/10.1007/s00018-020-03625-5

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  • DOI: https://doi.org/10.1007/s00018-020-03625-5

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